Traditional starter culture programs have depended on phage defense rotation strategies (PDRS) to minimize failures due to bacteriophage attack. See generally T. Klaenhammer, Advances in Applied Microbiology 30, 1 (1984); R. Lawrence et al., J. Dairy Res. 43, 141 (1976); H. Whitehead and G. Hunter, J. Dairy Res. 15, 112 (1947). When lytic phage appear in whey samples during defined single strain rotations, a replacement strain which is ideally phage-unrelated is incorporated into the rotation. History has proven, however, that it is difficult to identify sufficient numbers of phage-unrelated strains to run complex rotation programs. Secondly, few strains are available that remain insensitive long enough to make their introduction into factories worthwhile. See, e.g., A. Huggins, Food Technol. (June 1984).
There are additional problems with traditional starter culture rotation strategies. Although some stains are not attacked by existing phages when introduced, phage may eventually appear due to phage mutation, modification, and build-up. See H. Heap and R. Lawrence, N.Z.J. Dairy Sci. Technol. 11, 16 (1976); G. Limsowtin and B. Terzaghi, N.Z.J. Dairy Sci. Technol. 11, 251 (1976); L. Pearce, N.Z.J. Dairy Sci. Technol. 13, 166 (1978); M. Sanders and T. Klaenhammer, Appl. Environ. Microbiol. 40, 500 (1980). Moreover, in many cases, the longevity and starter activity of complex strain rotations is unpredictable and often leads to early failure. See, e.g., G. Limsowtin et al., N.Z.J. Dairy Sci. Technol. 13, 1 (1977); R. Thunell et al., J. Dairy Sci. 64, 2270 (1981). Furthermore, prolonged rotations involving numerous strains increase the level and diversity of phage contaminating the plant. Seek e.g., H. Heap and R. Lawrence, N.Z.J. Dairy Sci. Technol. 12, 213 (1981); R. Lawrence et al., J. Dairy Sci. 61, 1181 (1978); R. Thunell et al., J. Dairy Sci. 64, 2270 (1981).
Studies on phage-insensitive strains of lactococci have uncovered the genetic basis for a variety of different phage defense mechanisms. See T. Klaenhammer, FEMS Microbiol. Rev. 46, 313 (1987); M. Sanders, Biochemie 70, 411 (1988). Such phage defense mechanisms include prevention of adsorption (Ads), restriction and modification (R/M), and abortive infection (Hsp). Development of gene transfer systems in bacteria, such as conjugation, transformation, and electroporation, have provided the opportunity to construct strains with improved characteristics. The existence of conjugal transfer (Tra.sup.+) and phage resistance (Hsp or R/M) determinants on plasmids such as pTR2030 (Tra.sup.+, Hsp.sup.+, R.sup.+ /M.sup.+), pTN20 (Tra.sup.+, R.sup.+ /M.sup.+), and pAJ1106 (Tra.sup.+, Hsp.sup.+) facilitates construction of phage-resistant starter strains using simple conjugal strategies which are acceptable as a natural means by which to genetically manipulate food grade microorganisms. D. Higgins et al., J. Bacteriol. 170, 3435 (1988); A. Jarvis et al., Appl. Environ. Microbiol. 55, 1537 (1989); M. Sanders et al., Appl. Environ. Microbiol. 52, 1001 (1986); W. Sing and T. Klaenhammer, Appl. Environ. Microbiol. 51, 1264 (1986). When employed repeatedly in the field, transconjugants carrying pTR2030 (Hsp+) have survived prolonged use in milk fermentations. See M. Sanders, Biochemie 70, 411 (1988). However, phage resistant to the Hsp mechanism have been detected, and pose a viable threat to prolonged use of a single Hsp.sup.+ strain in rotation.
With neither traditional phage rotation strategies nor more recent genetic strategies providing a complete solution to the problem of phage infection of culture media, their is an ongoing need for new means for combating phage infections in the fermentation industry. The present invention is based on our ongoing research in this field.